A novel and simple procedure for the controlled-rate cryopreservation of peripheral blood progenitor cells (PBPCs) was introduced. A freezing bag housed in a protective aluminum canister was placed on top of a styrene foam box in the -85˚C electric freezer. A second set of samples was kept in cryotubes placed in a double styrene foam box in the same electric freezer. Measurement of the freezing rate in the PB bags and cryotubes demonstrated that this simple method for PBPC cryopreservation provided optimal conditions for both large-scale and small-scale cryopreservation. Within several days after autologous peripheral blood stem cell transplantation, we thawed the cells in the small sample tubes and evaluated the cell viability, the cell recovery, and the recovery rates of hematopoietic progenitor cells (HPCs), such as CD34 + cells and colonyforming unit-granulocyte/macrophage (CFU-GM) colonies. The median duration of cryopreservation was 59 days (range, 14 -365 days). According to our analysis, infusions of more than 2 × 10 6 CD34 + cells/kg body weight and 0.5 × 10 6 CFU-GM colonies/kg body weight after thawing had favorable influences on the neutrophil engraftment. We have therefore established a simple freezing method for cryopreservation of human PBPCs, which ensures the transplantability of hematopoietic progenitors even after thawing. In vitro HPC assay after thawing is important to evaluate the quality of cryopreservation procedures.peripheral blood progenitor cell; CD34 + cell; CFU-GM colony; cryopreservation

Total nucleated cell (TNC) count, CD34(+) cell count, colony-forming unit-granulocyte-macrophage (CFU-GM) content, and cell viability impact the outcome of umbilical cord blood (UCB) transplantation. Assessments of unit quality have usually been provided by cord blood banks (CBBs), but it is unclear whether pre-freezing tests or pre-transplant release tests performed by CBBs are reproducible. The aim of this study was to compare the UCB characteristics analyzed at the site of infusion of the UCB with those provided by CBBs. Samples were taken from 54 UCB units for assessment of post-thaw characteristics. TNC counts and CD34(+) cell contents measured at our hospital before infusion showed good correlations with values assessed in pre-freezing tests (r = 0.900 and 0.943, respectively) and pre-transplant release tests (r = 0.829 and 0.930, respectively). Our data reveal that the TNC counts and CD34(+) cell contents determined by pre-freezing and pre-transplant release tests, which are the most important UCB unit selection criteria, accurately reflected the quality of infused UCB units. However, CFU-GM content was poorly correlated (r = 0.560 and 0.606). Correlation of post-thaw cell viabilities measured before infusion and during the pre-transplant release tests was also poor (r = 0.308). We suggest that the TNC count and CD34(+) cell content estimated before cryopreservation and in pre-transplant release tests provided by CBBs are reproducible and can assist the transplant physicians in selection of appropriate UCB units.

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The aim of this study was to evaluate how a simple method of cryopreservation influences the quality of CD34+ cells in umbilical cord blood (UCB). The cells were dispensed into a double-compartment freezing bag, cryopreserved at -85 degrees C without a rate-controlled programmed freezer, and stored in the liquid phase of nitrogen. The viability of the CD34+ cells before freezing and after thawing was assessed by flow cytometry with 7-aminoactinomycin D and by colony-forming assays. Twenty UCB units cryopreserved for a median of 92 days were analyzed. Mean CD34+ cell viabilities before freezing were 99.8% +/- 0.4% and after thawing were 99.5% +/- 0.8% in large chambers, 99.6% +/- 0.5% in small chambers, and 99.4% +/- 0.6% in sample tubes. The mean values from colony-forming assays of the viable CD34+ cells before freezing were 30.7 +/- 6.8 (colony-forming units-granulocyte-macrophage [CFU-GM] per 100 viable CD34+ cells) and 68.5 +/- 14.8 (total CFUs per 100 viable CD34+ cells). The CFU-GM and total CFU values after thawing were, respectively, 32.7 +/- 9.0 and 66.0 +/- 13.4 in large chambers, 32.4 +/- 8.1 and 64.5 +/- 16.1 in small chambers, and 30.9 +/- 5.4 and 64.7 +/- 12.4 in sample tubes. The results of the colony-forming assays before freezing and after thawing were not significantly different. Our findings overall indicated that our simple method for the cryopreservation of UCB cells without a rate-controlled programmed freezer does not impair the clonogenic capacity of UCB progenitor cells. This cryopreservation method could provide cellular products adequate for hematopoietic stem cell transplantation.

A simple method for cryopreservation of CB cells without a rate-controlled programmed freezer could provide a sufficient-enough potential for the transplantability of HPCs after thawing.